1. Field of the Invention
This invention relates to assay methods for detecting inhibitors of farnesyltransferase enzymes. In particular, the invention relates to methods for detecting inhibitors using K-rasB preparations as the farnesyl acceptor substances.
2. Description of the Related Art
In recent years, some progress has been made in the elucidation of cellular events lending to the development or progression of various types of cancers. A great amount of research has centered on identifying genes which are altered or mutated in cancer relative to normal cells. In fact, genetic research has led to the identification of a variety of gene families in which mutations can lead to the development of a wide variety of tumors. The ras gene family is a family of closely related genes that frequently contain mutations involved in many human tumors, including tumors of virtually every tumor group (see, e.g., Bos, 1989). In fact, altered ras genes are the most frequently identified oncogenes in human tumors (Barbacid, 1987).
The ras gene family comprises three genes, H-ras, K-ras and N-ras, which encode similar proteins with molecular weights of about 21,000 (Barbacid, 1987). These proteins, often termed p21.sup.ras, comprise a family of GTP-binding and hydrolyzing proteins that regulate cell growth when bound to the inner surface of the plasma membrane (Hancock, et al., 1989). Overproduction of P21.sup.ras proteins or mutations that abolish their GTP-ase activity lead to uncontrolled cell division (Gibbs et al., 1989). However, the activity of normal and oncogenic Ras proteins requires their attachment to the inner leaflet of the plasma membrane. This process is initiated by the covalent attachment of a hydrophobic farnesyl group to a cysteine at the fourth position from the COOH-terminus of the Ras protein. Mutation of this cysteine to a serine prevents farnesylation, abrogates membrane attachment, and abolishes the transforming ability of oncogenic Ras proteins (Hancock et al., 1989; Schafer and Rine, 1992).
Four Ras proteins, designated H-Ras, N-Ras, K-rasA, and K-RasB, are expressed in animal cells (Barbacid, 1987). K-rasA and K-rasB are alternatively spliced products of a single gene (Barbacid, 1987). These proteins resemble each other closely with the exception of their COOH-terminal domains. All of them are believed to function similarly in activating cell growth. All four terminate in CAAX boxes with the following sequences: CVIM (K-rasB), CIIM (K-rasA), CVVM (N-Ras), and CVLS (H-Ras). K-rasB differs from the other three forms of Ras because it contains a prominent string of lysine residues (8 lysines among the 10 residues immediately adjacent to the farnesylated cysteine). These lysines assist in the membrane attachment of farnesylated K-rasB, presumably by binding to negatively charged phospholipids on the inner surface of the plasma membrane (Hancock et al., 1990). H-Ras is the form most often studied experimentally in cell culture. However, mutations in K-RasB are by far the most frequent in human tumors (Barbacid, 1987).
Farnesylation of Ras proteins is catalyzed by a heterodimeric enzyme, CAAX farnesyltransferase (farnesyltransferase), which was first purified and cloned from rat brain (Example I herein) and subsequently purified from bovine brain (Pompliano et al., 1993). This Zn.sup.2+ -containing enzyme catalyzes the Mg.sup.2+ -dependent transfer of a farnesyl group from farnesyl pyrophosphate (FPP) to Ras proteins and several other proteins, including nuclear lamins (Reiss et al., 1992).
CAAX farnesyltransferase is one of two enzymes that attach isoprenes to cysteines at the fourth position from the COOH-terminus of proteins (Brown and Goldstein, 1993; Casey, 1992). The second enzyme, CAAX geranylgeranyltransferase, is also known as geranylgeranyltransferase type-I or geranylgeranyltransferase-1. This enzyme transfers a 20-carbon geranylgeranyl group, which is even more hydrophobic than the 15-carbon farnesyl. Both prenyltransferases recognize COOH-terminal tetrapeptides that are designated as CAAX boxes in which C is cysteine, A stands for an aliphatic amino acid, and X is a variable amino acid that dictates the relative specificity of the protein for the two prenyltransferases. In most farnesylated proteins, including Ras proteins and nuclear lamins, X is methionine or serine. Geranylgeranylated proteins, including the GTP-binding protein Rap1B and the .gamma.-subunits of heterotrimeric G proteins, usually terminate in leucine (Brown and Goldstein, 1993; Casey, 1992; Reiss et al., 1992; Moores et al., 1991).
CAAX farnesyltransferase and geranylgeranyltransferase-1 are both .alpha./.beta. heterodimers (Brown and Goldstein, 1993; Casey, 1992; Moomaw and Casey, 1992; Yokoyama and Gelb, 1993). The .alpha. subunits of the two enzymes are identical (Example III herein, Seabra et al., 1991; Zhang et al., 1994), and the two rat .beta. subunits show 28% identity (Zhang et al., 1994). Genetic studies in yeast confirm that the .alpha.-subunits of the two enzymes are the product of the same gene (Schafer and Rine, 1992). So far it has not been possible to separate the .alpha. and .beta. subunits without denaturation, nor is it possible to produce high levels of one without the other by overexpression in animal (Chen et al., 1991b) or Sf9 cells (Chen et al., 1993; James et al., 1993). The .beta.-subunit of rat brain farnesyltransferase binds the CAAX substrate, as determined from cross-linking studies (see Examples, Reiss et al., 1991). The role of the .alpha.-subunit has not yet been delineated (Andres et al., 1993).
The existence of a shared .alpha.-subunit suggests that the two CAAX prenyltransferases may have some overlapping substrate specificity. Consistent with this notion, studies with partially purified geranylgeranyltransferase-1 showed that its substrate specificity overlaps that of farnesyltransferase. Yokoyama et al. (1991) showed that geranylgeranyltransferase-1 will transfer .sup.3 H!geranylgeranyl to a peptide corresponding to the COOH-terminal 10 residues of lamin B, which terminates in serine, albeit at much lower efficiency than was observed for leucine-terminated peptides. Moreover, the enzyme was able to attach .sup.3 !farnesyl as well as .sup.3 H!geranylgeranyl to peptides that terminate in leucine. In yeast, Trueblood et al. (1993) showed that the consequences of a deletion mutant of the .beta.-subunit of CAAX farnesyltransferase could be overcome partially by overexpression of the .beta. subunit of the geranylgeranyltransferase-1, and vice versa.
Although it appears to be clear that prenylation is a key event in ras-related cancer development, the nature of this event has remained obscure. Little is known, for example, of the role of farnesyltransferase enzyme involved in ras tumorigenesis or required by the tumor cell to achieve prenylation. If the mechanisms that underlie farnesylation of cancer-related proteins could be elucidated, then procedures and even pharmacologic agents could be developed in an attempt to control or inhibit expression of the oncogenic phenotype in a wide variety of cancers.